Numerous reports have documented changes in the amounts or functional activity of G proteins in cardiac pathology and with aging. Given the potential clinical impact of these observations it is critical that we gain a clear understanding of the normal signal transduction pathways. The studies outlined in this proposal will provide a framework for understanding what clinical significance these changes may have and may help in devising clinically useful interventions. The goal of this proposal is to use targeted mutagenesis of specific G protein genes as a tool to investigate cardiac muscarinic and adenosine receptor-G protein signaling mechanisms. Acetylcholine and adenosine are two important inhibitory modulators of cardiac performance and may have cardioprotective effects during periods of ischemia. Using mouse embryonic stem (ES) cells a panel of mutant lines will be generated in which one or more of the G protein subunits (alpha, beta, or gamma) is inactivated. The effects of these inactivations on receptor signaling will then be evaluated using in vitro differentiated ES cells as a model for cardiocytes. Our specific goals are to identify the G protein subunits required by each receptor for signaling to a common effector and by a single receptor for signaling to two different effectors (the atrial-type inwardly rectifying K+ channel and the ATP sensitive K+ channel). Chronotropic responses will also be monitored as an indicator of overall receptor signaling efficiency and will provide a physiological correlate for our single channel studies. Although I have had some training in cell physiology and metabolism with my PhD, to become an independent investigator, it is critical that I obtain extensive training in genetic and molecular techniques. After a five year hiatus during which I completed clinical training, I joined Dr. Richard Mortensen's laboratory to gain hands-on experience with molecular biology. To complete the studies described in this research plan, training in electrophysiology (under the guidance of Dr. Peter Vassilev) is also required. I have a very strong advisory committee of three experienced senior scientists, Dr. Gordon Williams, Dr. Christine Seidman, and Dr. Eva Neer, who will provide an invaluable resource for my scientific and personal career development. Long term I plan to use information gained from the studies outlined in this proposal to further investigate the role that G proteins play in cardiac disease and to characterize factors (metabolic, hormonal, and pharmacologic) that regulate expression and function of theses proteins. The ultimate objective is to be able to predictably and specifically modulate the functions of G proteins in a clinically beneficial manner.